Blood coagulation involves the assembly of calcium- dependent macromolecular complexes on phospholipid membranes. The penultimate step in blood coagulation is the conversion of prothrombin to thrombin, which is then responsible for the conversion of fibrinogen to fibrin. Uncontrolled or excessive thrombin generation leads to thrombosis, a major cause of morbidity and mortality. The protein C anticoagulant pathway is an essential mechanism for regulating thrombin formation, whereby activated protein C (APC) in association with its cofactor protein S enhances the inactivation of factor Va (FVa) and factor VIIIa (FVIIIa). Protein S, is a vitamin K-dependent protein that possesses both APC-dependent and APC-independent anticoagulant activity. The APC-independent activity of protein S has been ascribed to its ability to inhibit the "prothrombinase" and "tenase" procoagulant complexes. The major goals of this research proposal are to elucidate the structural and/or functional significance of the N-terminal domains of protein S including: the Gla domain, the thrombin sensitive region and the first epidermal growth factor domain. Initially, we will characterize the Gla domain of human protein S, PS(1-45), in the APC-independent mechanism of anticoagulation. PS (1-45) will be chemically synthesized and its calcium binding, phospholipid binding and functional properties investigated. We will then evaluate the ability of this domain to inhibit the FVa- and FX- dependent prothrombinase activity. Finally, we will determine the three-dimensional structure of PS(1-45) in the presence and absence of calcium using NMR spectroscopy. We believe that a comparison between these two structures will identify residues that are critical for calcium binding and thus involved in the calcium-dependent conformational perturbation. The other major goal of this proposal involves the APC-dependent cofactor activity of protein S, which requires the thrombin sensitive region (TSR) and first epidermal growth factor domain (EGF1). Initially, we will characterize the structural and functional properties of recombinantly expressed TSR-EGF1 (rhTSR-EGF1), to ensure this protein is properly folded. Simultaneously, we will uniformly 15N and 13C label rhTSR-EGF1 allowing us to determine the three-dimensional structure of TSR-EGF1 using heteronuclear NMR spectroscopy. We will then investigate the protein-protein interaction between rhTSR-EGF1 and APC or FXa using fluorescence spectroscopy, isothermal calorimetry and NMR spectroscopy. This structural information will identify the critical epitopes involved in complex formation and provide valuable information necessary to understand the mechanisms involved in regulating thrombus formation, which is of considerable clinical and basic science interest. New insights into these mechanisms may identify novel methods for the intervention in pathologic thrombus formation.